The phosphorylation and dephosphorylation of tyrosine residues in proteins and enzymes is crucial to many cellular signaling pathways in both health and disease. In vivo, tyrosine phosphorylation is dynamic and tightly controlled by the concerted actions of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs). Although the importance of tyrosine phosphorylation has been recognized for many years, the tools necessary to probe the phosphorylation and dephosphorylation of key tyrosine residues in important signaling proteins have been slow to develop. In this exploratory/developmental research project, we will create a new set of chemical probes of tyrosine phosphorylation. These probes will be incorporated into living cells and used to image tyrosine phosphorylation and dephosphorylation in vivo. The long-term objectives of this project are to establish a novel technology for real-time analysis of tyrosine phosphorylation in vivo and demonstrate its utility in the investigation of cell signaling under physiological and pathological conditions. To this end, we present the following SPECIFIC AIMS: (1) Develop and validate a series of novel peptide-based probes for PTP/PTK activity in vitro, (2) Illustrate the utility of these novel probes in imaging dynamic tyrosine phosphorylation/dephosphorylation processes in vivo. The research described in this proposal requires the unique combination of expertise in synthetic chemistry, biochemistry and cell biology embodied by the two Principal Investigators. Funding for this developmental project through the EBRG R21 mechanism will be instrumental in validating the new technology for imaging tyrosine phosphorylation and paving the way for future hypothesis-driven research on the importance of tyrosine phosphorylation in cellular signaling processes. RELEVANCE: Both tyrosine phosphatase and tyrosine kinase enzymes are important therapeutic targets for the treatment of many human diseases including diabetes, autoimmune disorders and cancer. In this research project we will develop a new technique for investigating the activity of these enzymes in live cells and in real time. This technique will help us to learn more about these enzymes in both health and disease, and ultimately facilitate the design of novel therapeutics. [unreadable] [unreadable] [unreadable]